This research was supported by practical research for innovative cancer control from the Japan Agency for Medical Research and Development (AMED). expression in cultured oesophageal keratinocytes is high, AKT2 and we could not show the difference between OSCC cells and normal oesophageal keratinocytes regarding the cytotoxicity with EGFR(2R)-lytic hybrid peptide in our experiments. Alternatively, the safety of EGFR(2R)-lytic hybrid peptide for the normal oesophagus was investigated by and organotypic 3D-culture experiments. We showed that EGFR(2R)-lytic hybrid peptide had a higher cytotoxicity than the lytic peptide fragment. According to the report of Papo the lytic peptide fragment forms a random coil structure in a solution, in which its ability to cause cell membrane disruption is weak21. However, the form of lytic peptide can be changed to an -helical structure when it is attracted to the cell surface by static electricity due to the lipid bilayer22,23 and it exerts enhanced cytotoxicity with cell membrane disruption21. Notably, the EGFR expression level on the cell surface affects the cytotoxicity of EGFR-lytic hybrid peptide15, suggesting that the EGFR-binding peptide fragment acts as an anchor to EGFR-expressing cells, and binding of the EGFR-binding fragment with EGFR on the cell surface contributes to change the lytic peptide fragment structurally and increase membranolytic cytotoxicity. Indeed, EGFR(2R)-lytic hybrid peptide showed high-level cytotoxicity against OSCC cells, whereas it was subtle when EGFR-binding peptide and lytic peptide fragments were not hybridized (co-administration of EGFR-binding peptide and lytic peptide fragments). These results indicate that the hybridisation of EGFR-binding peptide and lytic peptide fragments plays a key role to enhance the membranolytic cytotoxicity of lytic peptide fragments. The therapeutic effect of existing EGFR-targeting therapy on ESCC is not sufficient. In OSCC, EGFR is frequently expressed9, while the mutation rate is very low (1.1%)24. On the other hand, gene mutations and amplifications Chromocarb of EGFR downstream signalling pathways are frequently noted (78.6%)24. The therapeutic effect of Chromocarb existing EGFR-targeted therapies is achieved by blocking EGFR signalling in the tumour. Therefore, it is influenced by gene alteration of EGFR as well as EGFR downstream signal cascades. For example, in non-small lung cancer, response rates of EGFR-TKI are more favourable in patients with than without EGFR Chromocarb mutations25. Moreover, in colon cancer, the therapeutic effects of anti-EGFR antibody are weaker in patients with mutations of molecules downstream of EGFR than those in patients without such mutations26,27. These results suggest that the low response rate to existing EGFR-targeted therapies in OSCC patients might be due to the low frequency of EGFR mutation as well as high frequency of gene alteration of EGFR downstream signalling pathways. In this study, the anti-tumour effect of EGFR(2R)-lytic hybrid peptide is considered to depend on cell membranous EGFR expression, but not on the intracellular EGFR signalling cascades, because the pretreatment of OSCC cells with Erlotinib did not affect the cytotoxicity of EGFR(2R)-lytic hybrid peptide (Supplementary Fig. S3). Taken together, we believe that EGFR-targeted therapy using EGFR(2R)-lytic hybrid peptide is a valid strategy against OSCC. In this study, EGFR(2R)-lytic hybrid peptide induced rapid disintegration of the cell membrane and ATP depletion in OSCC cells. Cell membrane damage with LDH leakage indicates necrotic cell death28, whereas ATP depletion indicates the loss of functional integrity of living cells29. Although our data could not determine whether cell membrane disintegration precedes or follows.